Three Aedes species infested by mermithids in France

Mermithid nematodes (Nematoda, Mermithidae) parasitising mosquitoes could be useful as biological agents for the control of host populations. Nine female mosquitoes belonging to the species Aedes cantans, Ae. communis, and Ae. rusticus were found parasitised by mermithids in Northern France. Sequencing of partial 18S rDNA showed 100% sequence homology for all processed specimens. The mermithid sequences were closely related to specimens previously recorded from Anopheles gambiae in Senegal. However, 18S sequences do not allow for identification of nematodes at the genus or species level. Our specimens could also be related to Strelkovimermis spiculatus, or belong to other genera not yet sequenced such as Empidomermis, the only mermithid genus recorded from mosquitoes in France.


Introduction
Mermithids nematodes are obligate endoparasites of Arthropoda, especially insects. They are considered host specific, usually at the host-species or at the family-species level [17]. Species parasitising mosquitoes have probably been the most studied, as they can be used as biological agents to control their host populations [1,6,15,16]. The presence of mosquito mermithids in France is poorly known, except for the description of a new species isolated from Aedes detritus in Southern France [7]. During a research program related to mosquito arboviruses, mosquitoes were sampled in Northern France. We report here the presence of mermithids in three different mosquito species analyzed by partial 18S ribosomal DNA sequencing.

Mosquito sampling
During a three-year epidemiological program focusing on mosquito arboviruses, mosquito larvae were episodically collected to create a qualitative inventory and assess the feasibility of establishing laboratory colonies of local mosquitoes for vector competence experiments. To this end, mosquito larvae were sampled in 2019 in two selected localities in north-eastern France: Berru on April 1st (49.267533 N, 4.133583 E) and in the vicinity of the Der-Chantecoq Lake on April 16th (48.576553 N, 4.692353 E).
Water puddles located in sylvatic environments were sampled by hand using a deeper. Collected larvae were placed in jars containing water from the local environment, then immediately transported to the insectary and placed in two labelled cages before emergence of adults. Larvae were fed yeast pellets and maintained at 22°C. Adults were maintained at 22°C, 60% relative humidity and given free access to a 10% sucrose Edited by: Jean-Lou Justine *Corresponding author: j.martinet.p@gmail.com solution. Each population was monitored daily in the laboratory. Mermithid infestation was characterized by the emergence of parasites escaping from adult mosquitoes.

Mosquito processing
Emerged mosquitoes were anesthetized by cold, and morphologically identified at the species level using the MosKeyTool taxonomic key [9].

Mermithid processing
The anterior and posterior parts of each worm were cut off and cleared in Amman lactophenol between the slide and cover slide. These specimens are available upon request to the authors. Pictures were taken using Stream Essentials Ó software version 1.7 and a DP-26 video camera connected to a SZX10 stereomicroscope (Olympus, Tokyo, Japan).
Genomic DNA was extracted from the middle part of the worm. Molecular identification of mermithids was performed by amplification and sequencing of partial 18S rDNA thanks to cycles and primers Merm forward 5 0 -CAAGGACGAAAGT TAGAGGTTC-3 0 and Merm reverse 5 0 -GGAAACCTTGT-TACGACTTTTA-3 0 as proposed by Kobylinski et al. [12].

Molecular analysis
Amplicons were sequenced through Sanger technology (Genewiz, Leipzig, Germany). First, mosquito and mermithid sequences (Table 1) were compared to existing GenBank sequences with the BLAST algorithm [2] and mosquito identification was considered accurate when similarity was higher than 99%. Second, mermithid sequences were edited and aligned using Muscle software [8]. The GTR+G model of molecular evolution was determined with ModelTest-NG [5] and the phylogenetic tree was constructed using the maximum likelihood (ML) method in MEGA11 [20].

Mosquitoes
Mosquito larvae collected in Berru included 45 females (12 Aedes cantans, 1 Ae. communis and 32 Ae. rusticus). Samples collected in the Der-Chantecoq lake included 55 Ae. rusticus females. As the first evidence of parasitism was observed on female mosquitoes at a time when males were already discarded from the cages, we do not possess data concerning Mermithid infestation in male mosquitoes.
Five female mosquitoes from Berru were infested by mermithids: four Ae. cantans and one Ae. communis.
Four female mosquitoes from the Der-Chantecoq lake were infested by mermithids (Fig. 1).
The identification of the infested mosquitoes was based upon morphological characters and by sequencing showing more than 99.6% of similarity with the reference sequences

Mermithids
Observation of the parasite juvenile stages revealed a posterior part with a straight spur (Fig. 1).
Sequences of 776 bp were obtained on the processed specimens. We analyzed an alignment of 750 bp of partial 18S rDNA sequences in order to compare our sequences with homologous ones available in GenBank. The sequences obtained from the mermithids isolated from the nine infected females were all identical (100% homology: no variability observed in the specimens processed in the present study). They are available in GenBank under accession numbers OQ249533-OQ249544.
The BLAST analysis showed that the closest sequence is that of a Mermithidae sp. isolated in some Anopheles gambiae from Senegal (99.21% homology with sequence KC243312 obtained by comparison of 756 bp of JOSN1 showing 6 variable and 750 conserved positions out of a total of 756 compared nucleotides) followed by several sequences of Strelkovimermis spiculatus (95.67% homology, meaning 729 conserved and 33 variable positions out of a total of 762). The ML tree obtained is shown in Figure 2.
We were, however, unable to amplify the COI domain of the mermithids with both published and in-house designed primers.

Discussion
Reliable morphological identification of mermithids must be performed on adults which constitute a free stage of these parasites. Unfortunately, we were not able to observe such stages and thus could not reliably identify the parasites collected. According to Nickle, they could belong to several genera (Reesimermis, Perutilimermis) [14].
According to 18S rDNA sequences, the specimens most closely related to ours were those isolated from Senegalese malaria vector mosquitoes Anopheles gambiae [12], which remained unidentified according to i) the difficulty in identifying parasites stages using morphological characters, and ii) the lack of a match with other sequences available in GenBank.
The specimens we collected and processed exhibited 6 mutations (99.21% homology) when compared to these Senegalese mermithids (GenBank accession number KJ636371). Considering that 18S rDNA is a highly conserved molecular marker, we cannot conclude regarding the exact identification of our samples at a species nor genus level. Studies carried out on triatomine bugs [4] as well as one study carried out on the digenean Fasciola hepatica [3], estimated that the conventional molecular clock rate is 1.8 Â 10 À10 substitutions per site per year (1.8% per 100 my) for the evolution of the 18S gene. If this calibration is accurate, the divergence time between Senegalese and French mosquito specimens would be close to 100 million years. Unfortunately, we were not able to amplify mermithid COI mtDNA, despite several repeats and the use of published and in-house designed primers. Nonetheless, based on the 100% homology of sequences between our samples, we can only conclude that they should belong either to the same species or to a very closely related one. The absence of Empidomermis, Culicimermis, Hydromermis, or Perutilimermis 18 S rDNA sequence entries in GenBank does not allow reliable identification down to the species level.
To date, Strelkovimermis samples (closest species identified in GenBank with 95.67% homology with our samples) have never been collected in France. To our knowledge, the only available report of a mermithid nematode in French mosquitoes is related to the original description of Empidomermis riouxi Doucet, Laumond & Bain, 1979 from Aedes detritus in Southern France [7]. We cannot exclude, based on our results, that the specimens processed in the present study could belong to this species.
The paucity of available data prohibits positive identification of the parasites we processed as previously encountered in the mermithid parasitism of bees or black flies [19,21]. Considering the importance of these nematodes in the biocontrol of mosquitoes, our work will, however, provide some information for future investigations on mermithids. Repeated and largescale use of current vector control strategies based on long lasting insecticide net distribution and indoor residual spraying of insecticides has led to an increased prevalence of mosquito resistance. Similarly, excessive insecticide use in agriculture has led to environmental pollution with an ecological impact on fauna and flora. New and innovative control strategies, such as the use of mermithids as biological agents to fight vectorborne diseases, remain to be explored.